J. Amer. Soc. Hort. Sci. 122(2):245-252. 1997.
Changes in Alternative Pathway and
Mitochondrial Respiration in Avocado in
Response to Elevated Carbon Dioxide Levels
Diana L. Lange1 and Adel A. Kader
Department of Pomology, University of California, Davis, CA 95616
ADDITIONAL INDEX WORDS.
Persea americana, alternative oxidase, cyanide-resistant
respiration, respiratory control, residual respiration
ABSTRACT.
Partially ripened avocado [Persea americana (Mill.) cv. Hass] fruit
harvested in either June or Aug. 1994 were kept at 10 °C in air (21% O2), 20%
CO2(17% O2, balance N2), or 40% CO2 (13% O2, balance N2) for 7 to 12 days and
then were transferred to air at 10 °C for 2 to 3 days. Mitochondrial respiration was
stimulated in response to elevated CO2 treatments at 10 °C. A shift to alternative
pathway (Alt) respiration occurred on day 4 in experiments using avocados from
both harvest dates, with a return to initial levels in only the 20% CO2-treated fruit
(June-harvested fruit after return to air). Elevated CO2 at 20 °C decreased the in
vitro O2 consumption of isolated mitochondria compared to mitochondria kept in
air. The Alt pathway contributed less to the total O2 uptake of CO2-treated
mitochondria compared to mitochondria kept in air. The respiratory control ratios
of the CO2-treated fruit and mitochondria were higher and lower, respectively than
the air controls. Induction of 33 to 37 kD proteins (corresponding to the size of
the alternative oxidase proteins) occurred in avocados after 4 days in 40% CO2.
These results indicate that elevated CO2 has various effects depending on
concentration, duration and temperature of exposure, and mitochondrial function
of avocado fruit, such as increased and altered respiratory oxidation and upregulation of alternative oxidase proteins.
The alternative respiratory (Alt) pathway, which is cyanide resistant, is present in a wide
range of plant tissues (Laties, 1982; Solomos, 1977; Solomos and Laties, 1976),
including avocados (Laties, 1982), cucumbers (Cucumis sativus L.) (Morohashi et al.,
1991), oranges [Citrus sinensis (L.) Osbeck] (Bruemmer, 1989), apples (Malus
domestica Borkh.), and peppers (Capsicum annuum L.) (Lurie and Klein, 1989). While
the function of this nonphosphorylating pathway is apparent in thermogenic species, its
role in nonthermogenic tissue is unclear (Mclntosh, 1994). The Alt pathway may provide
a way to disperse excess reducing power when cytochrome oxidase is inhibited
Received for publication 5 Mar. 1996. Accepted for publication 19 Sept. 1996.Research supported in part by U.S.
Dept. of Agriculture research agreement no. 58-319R-3-004. The cost of publishing this paper was defrayed in part
by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked
advertisement solely to indicate this fact.
1
Extension specialist; current address, Dept. of Horticulture, Southwest Purdue Agricultural Research and Extension
Center, R.R. 6, Box 139A, Vincennes, IN 47591.
(Lambers, 1982). Collier and Cummins (1991) reported that the Alt pathway may have
been used during Saxifraga cernua L. petal unfolding as an inefficient energy source.
During fruit ripening, the energy charge of the tissue is high and the rate of cytochrome
(Cyt) pathway oxidation is coupled tightly to oxidative phosphorylation. The Alt pathway
may provide a means of oxidizing the large electron flow that occurs during fruit ripening
without producing as large amounts of ATP as occurs when the Cyt pathway is
operating alone.
The ability of CO2 to stimulate the Alt pathway has been reported previously, but most
of these studies focused on the effects of CO2 in combination with ethylene (Day et al.,
1978; Laties, 1987). Laties (1982) concluded that CO2 appeared to enhance ethyleneinduced Alt respiration. Carbon dioxide alone has sustained the cyanide resistant
pathway in wheat (Triticum aestivum L.) seedlings (McCaig and Hill, 1977), carnation
(Dianthus caryophyllus L.) callus (Palet et al., 1991), and Jerusalem artichoke
(Helianthus tuberosus L.) tubers (Stegink and Siedow, 1986). To our knowledge, the
ability of CO2 to stimulate the Alt pathway in fruit tissue has not been demonstrated.
One of our objectives was to determine if, and to what degree, CO2 stimulates the Alt
pathway.
Elthon and Mclntosh (1987) identified alternative oxidase (AltOx) proteins (molecular
weights = 35 to 37 kD) in the thermogenic spadix of Sauromatum guttatum Schott and
later developed monoclonal antibodies to these proteins (Elthon et al., 1989). AltOx
proteins subsequently have been identified in a number of non-thermogenic plant
species, including avocado fruit, etiolated mung bean (Vigna radiata L.) hypocotyls,
fresh potato (Solanum tuberosum L.) tubers, and tobacco (Nicotiana tabacum L.) callus
(Elthon et al., 1989). Rhoads and Mclntosh (1993) reported that increased Alt pathway
activity correlated with an accumulation of a 35-kD protein in tobacco suspension
cultures and de novo transcription and translation were necessary to cause the
maximum accumulation of the 35-kD protein. Yoshimoto et al. (1989) reported that the
Alt pathway was induced simultaneously with the appearance of a 36-kD protein in
yeast. Although the AltOx proteins are present in fruit tissue, there is still some question
of whether or not they are constitutive and always present or synthesized de novo in
coordination with developmental changes. AltOx in pear (Pyrus communis L.) was
constitutive, and changes in its activity did not involve transcriptional or translational
events (R. J. Romani, unpublished data).
Huang and Romani (1991) reported that avocado mitochondria have an intrinsic
homeostatic capacity in which disrupted energy-linked functions are self-restored. In
addition, the storage life of avocados can be extended by exposure to elevated CO2
atmospheres (Spalding and Reeder, 1974; Trüter and Eksteen, 1987).Therefore, we
used avocado fruit and their mitochondria to study the direct and indirect effects of CO2
stress on enhancement and partitioning of respiration in fruit tissue.
Materials and Methods
'Hass' avocado fruit were harvested at commercial
maturity (≥24% dry mass) from an orchard in Santa Barbara County, Calif., and were
transported to our laboratory in Davis, Calif., where they were stored at 10 °C for up to 1
week until initiation of the experiments. Fruit uniform in size and free from defects were
PLANT MATERIAL AND TREATMENTS.
selected. Before initiating experiments, fruit were partially ripened at 20 °C for 4 d with a
continuous flow of ethylene at 10 µL-L-1 in humidified air at a flow rate of 400 mL-min-1.
Experiments were conducted at 10 °C for up to 15 d. Nine fruit were placed in a 4-L
glass jar and ventilated with humidified air (21% O2 + 0.3% CO2 + balance N2) or a
specified gas mixture at a continuous flow rate of 100 mL-min-1. The gas mixtures
included 20% CO2 mixed with air (20% CO2+17% O2+balance N2) or 40% CO2 mixed
with air (40% CO2+ 13% O2+ balance N2). Avocado fruit were kept in air or CO2enriched atmospheres at 10 °C for 12 (June 1994) or 7 (Aug. 1994) d and then
transferred to air for 3 (June) or 2 (August) d.
Individual fruit were selected for each experiment based on screening for uniform C2H4
production rates. Partially ripened avocados were screened after 4 d of ripening. For
every sampling date, treatments were replicated three times, each replicate having
three fruit. All of the fruit for one sampling date for each treatment were contained in the
same jar.
Intact avocados were exposed to air or CO2-enriched atmospheres at 10 °C and then
were used in mitochondrial assays unless otherwise stated.
Avocado fruit mitochondria were
isolated using the method of Moreau and Romani (1982b). All extraction procedures
were performed over ice. Homogenization of the fruit tissue was conducted in a 10°C
storage room. For the mitochondrial O2 uptake assays, 100 g of tissue were
homogenized in 300 mL isolation medium using a fine wire-mesh screen submersed in
medium. The isolation medium consisted of 0.25 M sucrose, 50 mM potassium
phosphate (pH =7.2), 5 mM EDTA, 5 mM ß-mercaptoethanol, 0.2% (w/v) soluble (40,000
MW) PVP, and 0.1% BSA. In all experiments, the homogenate was filtered through four
layers of cheesecloth and centrifuged at 2000 x g for 10 min. The supernatant was
filtered through four layers of cheesecloth and centrifuged at 10,000 x g for 15 min. This
supernatant was discarded, and the pellet was resuspended in 3 mL wash medium and
homogenized in the presence of 11 mL additional wash medium. The wash medium
consisted of 0.25 M sucrose, 50 mM potassium phosphate (pH = 7.2), 5 mM ßmercaptoethanol, and 0.1% BSA. This homogenate was recentrifuged at 2000 x g for 5
min to pelletize remaining chloroplasts out of the mitrochondrial supernatant.
MITOCHONDRIAL ISOLATION AND PARTIAL PURIFICATION.
Partial purification of the mitochondria was conducted in the following manner. An 8,000
x g centrifugation step was substituted with a 20,000 x g centrifugation step through 20
mL of 25% Percoll (Sigma Chemical Co., St. Louis) "pad" for 10 min [as described in
Day and Hanson (1977) and modified by Romani (1994) personal communication]. This
partial purification was necessary to eliminate most of the nonmitochondrial organelles
that would interfere with the oxidase assays. A full Percoll purification system was not
used (Moreau and Romani, 1982a) because a more rapid cleanup was necessary. The
pad medium consisted of 25% Percoll, 0.25 M sucrose, and 50 mM potassium
phosphate (pH = 7.2). The resulting mitochondrial pellet was washed with wash medium
and centrifuged at 2000 x g for 5 min several times to remove the Percoll with the
supernatant, and then the mitochondrial pellet was resuspended in 0.3 to 0.5 mL wash
medium. Between the addition of wash medium and centrifugation steps, the
supernatant was removed by vacuum aspiration and discarded. Protein content of the
purified mitochondrial preparation was determined by the Bradford (1976) method using
BSA as the standard.
Mitochondrial
functions were monitored, including the measurement of O2 uptake, the contributions of
different oxidase pathways toward total O2 consumption, and respiratory control ratios
that indicate the degree of oxidative phosphorylation. One milliliter of reaction media
[0.25 M sucrose, 0.1% BSA, 50 mM phosphate (pH 7.2), 1 mM MgCl2, 10 µM CoA, 100
µM TPP (thiamin pyrophosphate), and 100 mM NAD+] and an appropriate aliquot of
mitochondria (0.2 to 0.4 mg
protein) were placed in a 1-mL
chamber fashioned from plexiglass
and equipped with an O2 electrode
(Yellow
Springs
Instruments,
Yellow
Springs,
Ohio).The
electrode was maintained at a
constant temperature of 25 °C. The
reaction mixture was constantly
stirred with a micro stir bar. The O2
content of air-saturated water was
estimated according to Estabrook
(1987). Oxygen consumption was
measured following the addition of
10 mM malate as substrate. The
contribution of the Cyt and Alt
pathways to total respiration was
determined using 1mM KCN and 3
mM
SHAM
(salicylhydroxamic
acid), respectively. KCN was
always added first to avoid
stimulation of O2 uptake by SHAM
through CN-sensitive peroxidases
(Møller et al., 1988). Respiratory
control
ratios
(RCR)
were
determined
by
adding
stoichiometric amounts of ADP
(≈0.15 mM) and measuring the
state 3:state 4 O2 consumption
rates ratio.
POLAROGRAPHIC MEASUREMENT OF O2 UPTAKE AND RESPIRATORY CONTROL.
IN VITRO EXPOSURE OF ISOLATED MITOCHONDRIA TO ELEVATED CO2 ATMOSPHERES. To
measure the effects of in vitro exposure to stress levels of CO2, mitochondria were
extracted as previously described from avocado fruit stored in air for <2 weeks at 10 °C
and were ripened partially with a continuous flow of C2H4 at l0 µL-L-1 in humidified air at
a flow rate of 400 mL-min-1. The fruit were ripened partially to facilitate tissue
homogenization. The mitochondria were suspended in reaction mixture and separated
into three equal aliquots (one per treatment) of 10 mL each in a 50-mL Erlenmeyer
flask. The mitochondrial suspensions were held at 20 °C on a rotary shaker on a low
setting. Each flask was sealed
with a serum cap supplied with
inlet and outlet flow lines to the
headspace. Air or the CO2enriched atmosphere (20% CO2 +
17% O2 or 40% CO2 + 13% O2)
was
flushed
through
the
headspace at 20 mL-min-1.
Before
each
assay,
the
headspace
was
aerated
momentarily to provide necessary
O2 as a substrate for oxidase
reactions. At the designated
times, a 1-mL aliquot of the
mitochondrial
mixture
was
removed
and
tested
for
respiratory control and different
oxidase pathway contributions.
DETERMINING
ALTERNATIVE
OXIDASE
(ALTOX)
PROTEIN
ABUNDANCE. Mitochondria were
isolated and partially purified as
previously described with minor
modifications. No BSA was used
in the isolation, wash, or
purification medium to avoid
overestimation of protein in the
samples.
Partially
purified
samples were dialyzed overnight
at 4 °C in 5 mM phosphate buffer
(pH 7.2) and 10% glycerol to
remove sucrose and salts, frozen
in liquid N2 and stored at -80 °C
for ≤5 months. Samples were
prepared as previously described
by Elthon and Mclntosh (1987)
with the following modifications: ß-mercaptoethanol concentration was increased to10%
(v/v), and the samples were boiled for 5 min before adding the tracking dye 0.04%
bromophenol blue. Electrophoresis was conducted with the buffer system of Laemmli
(1970) using a 5% stacking and a 15% polyacrylamide separating gel. The gels were
stained with Coomassie Blue to verify equal loading of protein. Bio-Rad low molecularmass protein standards, either unstained or biotinylated for gels or immunoblots,
respectively, were used to estimate molecular mass. Protein blotting followed the
protocol of Blake et al. (1984) except that antibody incubations were for 2 h at room
temperature. In accordance with Elthon and Mclntosh (1987), the AltOx proteins were
detected with a 1 monoclonal
antibody :10 AltOx dilution from
Sauromatum guttatum (courtesy
of T.Elthon).
Results and Discussion
The O2 uptake of mitochondria
extracted from partially ripe
'Hass' avocados that were kept at
10 °C in air (Fig. 1) followed a
similar pattern to that of the intact
fruit (Lange and Kader, 1996).
Uptake of O2 by mitochondria
from June-harvested fruit kept in
air gradually declined over the
15-d storage period at 10 °C,
whereas
O2
uptake
by
mitochondria
from
Augustharvested fruit peaked on day 4.
Avocado fruit exposed to 20%
CO2 had an increase in the O2
uptake of extracted mitochondria
on day 4 (Fig. 1 A and B). The O2
uptake of 40% CO2-stored
mitochondria
from
Juneharvested avocado fruit was 3fold greater than the air control
on treatment days 4 and 9 (Fig.
1A), whereas with Augustharvested fruit, mitochondrial O2
uptake was 2-fold greater than
the air control on day 4 (Fig. IB).
After transfer to air, the
mitochondrial O2 uptake of 20%
CO2- (Fig. 1A) and 40% CO2treated (Fig. 1A and B )
avocados
increased
again.
Mathooko et al. (1995) also found that elevated CO2 concentrations increased the
mitochondrial activity of cucumber fruit exposed to 30% or 60% CO2. Perez-Trejo et al.
(1981) found that only 2 to 3 h of exposure to 10% to 30% CO2 were required to cause
a 6-fold rise in respiration of potato tubers. This increased whole potato tuber respiration
may be a result of in-creased mitochondrial respiration. Lange and Kader (1997) found
that 40% CO2-stored avocado fruit had increased total fruit respiration, just as we
observed in this fruit study with mitochondrial respiration.
Mitochondrial respiration of Juneharvested, air-stored avocado fruit
was relatively low and gradually
declined, regardless of the pathway
contributing to O2 uptake (Fig. 2A).
In 20% CO2-stored avocado fruit,
the
primary
terminal
oxidase
pathway was the cytochrome
oxidase (Cyt) pathway on day 4.
However, on day 9, the predominant
terminal oxidase pathway was the
alternative (Alt) pathway (Fig. 2B).
Treatment of avocado fruit with 40%
CO2 elicited a similar effect as that of
20% CO2, but after transfer to air or
2 d, the Alt pathway became
predominant again (Fig. 2C). The
August-harvested avocados had a
similar pattern of response due to
terminal oxidase pathways with most of the differences occurring on day 4 (Fig. 3). The
Cyt pathway was favored in air-stored fruit (Fig. 3A), whereas the Alt pathway was
favored in avocados exposed to either CO2 concentration (Fig. 3 B and C). Residual
respiration (i.e., respiration in the presence of KCN and SHAM) usually accounts for at
most 10% to 20% of the total O2 uptake in fruit (Romani, personal communication). In
our study, residual respiration was substantial (often >30%) and appeared to best
stimulated by elevated CO2 exposure (Figs. 2 and 3).
Laties (1982) reported that exposure of potato tubers to 10% CO2 for 72 h yielded CNresistant tissue as well as a significant rise in residual respiration. Residual respiration,
at least in part, may be due to monoxygenases, which probably contribute to the total
O2 consumed by plant tissues (Day et al., 1980). There also may be a contribution of αoxidation to residual respiration.
The direct in vitro exposure to CO2 caused partial inhibition of O2 uptake of mitochondria
extracted from 'Hass' avocados (Fig.4). Within 2 h, the rates of O2 uptake of
mitochondria kept in air, 20% CO2, or 40% CO2 were all reduced by ≈50%. Rates
remained at a constant level in mitochondria kept in air, whereas the respiration rates of
CO2-treated mitochondria decreased to 20% of the initial rate after 20 h at 20 °C.
Mathooko et al. (1995) also found that elevated CO2 concentrations (up to 60%)
inhibited the in vitro O2 uptake rates of mitochondria from cucumber fruit, broccoli
(Brassica oleracea var. italica Plenck) buds, and carrots (Daucus carota L.).
Most of the initial O2 uptake of extracted avocado mitochondria was contributed by the
Alt pathway (Fig. 5), but this decreased to 50%, 40%, and 20% of the initial activity after
only 2 h in air, 20% CO2, and 40% CO2, respectively. For the rest of the experiment, the
mitochondria kept in air maintained an Alt O2 uptake that was 50% of the initial rate,
whereas the CO2-treated mitochondria had a continued decrease in O2 uptake resulting
from the Alt pathway. The relative contribution of the Cyt pathway was higher than the
other pathways in the CO2-treated
mitochondria after 2 and 4 h at 20
°C (Figs. 5B and 5C).
Since the CO2 treatments included
slightly lower levels of 02 (17% for
the 20% CO2 treatment and 13%
for the 40% CO2 treatment) than
the air treatment (21% O2), partial
inhibition of the Alt pathway by CO2
may be due to the higher Km of
alternative oxidase for O2, which is
estimated to be 10-times higher
than the Km or cytochrome oxidase
(Solomos, 1977). For example,
Sherald and Sisler (1972) observed
a Km of 11 to 14 µM for AltOx
compared with 1.2 to 1.4 µM for
CytOx. However, the levels of O2 in
the C02 treatments are too high to
have a major effect on the affinities
of these terminal oxidase pathways
for O2. Other direct effects of C02
on the mitochondria, such as the
acidification of the intracellular
spaces or structural changes in
proteins or lipid membranes, may
have played a role as well (RomoParada et al., 1991; Shipway and
Bramlage, 1973). Moriguchi and
Romani (1995) concluded that
exposure of avocado fruit to CO2rich atmospheres enhanced the
capacity of their mitochondria to
restore energy-linked functions.
The respiratory control ratio (RCR)
is defined as the state 3 O2 uptake
rate (in the presence of added ADP) divided by the state 4 O2 uptake rate (where ADP
supply has been depleted). RCR values for freshly isolated avocado mitochondria are in
the range of 2 to 5 (Moreau and Romani, 1982a). The indirect effect of CO2 on the RCR
of treated avocado fruit was to maintain or to protect the tissue from a loss of respiratory
control (Fig. 6). After transfer to air for 2 to 3 d, the RCR levels dropped to near 1,
regardless of the previous treatment. The effect of CO2 on the RCR levels of either
June- or August-harvested 'Hass' avocado fruit was similar. Elevated CO2 atmospheres
have slowed senescence of a wide range of horticultural commodities (Kader, 1986;
Wang, 1990).This slowing may be due partially to the protective role that CO2 may play
in maintaining membrane integrity (Day et al., 1978).
The RCR levels of in vitro CO2-treated
avocado mitochondria were slightly lower
than mitochondria kept in air after 2 and
4 h of CO, exposure (Fig. 7). RCR
dropped to 1 in all treatments, indicating
the total loss of respiratory control, after
20 h. (RCR =1). Although it was not a
strong uncoupler, the direct effect of CO2
appears to be through an uncoupling of
oxidative phosphorylation, as was
described previously by Shipway and
Bramlage (1973).
Western blot (immunoblot) analysis,
using a monoclonal anti-body to the
AltOx proteins, revealed the presence of
three or more proteins in the 33- to 41kDa range (Figs. 8-10). Elthon et al.
(1989) reported the presence of
alternative oxidase (AltOx) proteins in
avocado fruit (range = 35 to 37 kDa). The
Alt pathway is stimulated by ethylene
produced during climacteric fruit ripening
(Laties, 1982). Since partially ripe
avocado fruit tissue that was producing
large amounts of ethylene was used in
these experiments, there was already an
abundance of AltOx proteins on day 0
(Figs. 8 and 9) (Lange and Kader, 1996).
On days 9 and 15 (labeled AT-Air) of air
storage of June-harvested avocado fruit
at 10 °C, the AltOx proteins accumulated
to higher than initial levels (Fig. 8).
Exposure to CO2-enriched atmospheres
decreased the amount of AltOx proteins
until after transfer to air for 3 d at 10 °C,
after which there was no effect of
previous treatment with CO2 on the
abundance of AltOx proteins. In Augustharvested avocado fruit stored at 10 °C,
an accumulation of AltOx proteins only
occurred in fruit stored in 40% CO2 for 4
d (Fig. 9). Again, after transfer to air for 2
d, there were no differences in the levels
of AltOx proteins. In a second experiment
to determine the effects of all treatments
on August-harvested avocados on day 4
(Fig. 10), the air-stored fruit had small amounts of AltOx proteins, the 20% CO2-stored
fruit had intermediate levels, and the 40% CO2-stored fruit had a high abundance of
AltOx proteins.
Expression of the Alt pathway in
nonthermogenic tissues is often
correlated with increased metabolic
activity, such as that observed in fruit
ripening or wound stress responses
(Day et al., 1980; Laties, 1982;
Palmer, 1976; Solomos, 1977). One
reason for Alt pathway expression
during fruit ripening or stress
responses may be to allow for the
continued generation of synthetic
intermediates by the mitochondria
when the energy charge is high
(Lambers,
1982).
In
addition,
expression of the Alt pathway allows
for the recycling of mitochondrial
matrix and cytoplasmic NADH by bypassing normal respiratory control.
Possibly, the increased expression of the Alt pathway during elevated CO2 exposure
was due to a physiological response that triggered activation of the Alt path-way. During
conditions of Cyt pathway inhibition (such as the use of stress levels of CO2), the
fermentative and Alt pathways are stimulated. Induction of the Alt pathway, compared to
induction of the fermentative pathway, results in an 8-fold increase in ATP production,
less substrate consumption, less disturbance to normal metabolism by maintaining the
Krebs cycle, and, therefore, greater tolerance of the commodity to high CO2 stress.
Recently, the importance of the Alt pathway in vivo has become clearer with the advent
of transgenic tobacco plants that have reduced or over expressed AltOx (Vanlerberghe
et al., 1994). The antisense-AltOx tobacco suspension cells did not survive when they
were grown under conditions that inhibited the Cyt pathway, while the wild type cells
were able to grow due to the functioning of AltOx. Cells with over expressed AltOx did
not have increased partitioning to the Alt pathway, suggesting that this partitioning may
be subject to additional regulatory factors in vivo, such as post-translational
modifications to AltOx protein that limits AltOx pathway activity.
Mitz (1979) suggested that the mechanism of CO2 action in disrupting typical cellular
function may be due to changes in protein configuration and membrane permeability.
Taking into account these broad changes in plant cells, the use of stress levels of CO2
to control disease, insects, or physiological disorders in plant tissues may be a feasible
alternative to using chemicals, provided that specific plant tissues and treatment
conditions (time, temperature, relative humidity, and O2 and CO2 concentrations) are
thoroughly tested before applying these treatments.
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